General Discussion and Conclusion - Biochemical and physiological aspects of volume regulation

The first challenge of this study was to establish an optimal experimental system to investigate the control of cell volume in bull epididymal spermatozoa. In the majority of the preceding studies, epididymal sperm have been recovered into a medium isotonic with seminal plasma or even into seminal plasma itself (Dott et al. 1979; Schweisguth and Hammerstedt 1992;

Gwathmey et al. 2003; Jones et al. 2008). While this mimics to some degree the tonicity effect of ejaculation, allowing subsequent investigation of the way in which sperm may respond generally to osmotic stress (Yeung et al. 2002; 2004), it has an important flaw where physiological studies of epididymal maturation are intended. If the immature cells do not possess volume regulatory ability, they may be well compromised by the stress they encounter on mixing with medium of markedly lower tonicity than their in vivo environment. Very recently, water transport in murine epididymal sperm has been investigated after recovery into environment isotonic with epididymal plasma (Callies et al. 2008); however, there has been a lack of study on the volumetric behaviour of mature and immature epididymal sperm released into different osmotic systems. In the present study (Paper Ι), three different treatment systems were tested on caput and cauda epididymal spermatozoa. To standardize the starting material, all samples were recovered in a medium isotonic with cauda epididymal plasma (HBS-360 mOsm kg^-1). They were then incubated either in the same medium (no osmotic challenge) and or in a medium isotonic with seminal plasma (HBS-300 mOsm kg^-1, osmotic challenge), or in homologous seminal plasma (osmotic challenge similar to that encountered on ejaculation). After incubation, spermatozoa were exposed to HBS which was either isotonic or hypotonic relative to the primary incubation medium (for further details see Paper Ι, Fig. 3-1). Primary incubation in either HBS-360 or HBS-300 had no effect on the membrane integrity of cauda sperm; however, incubation in seminal plasma had a tendency to reduce membrane integrity. The membrane integrity of caput sperm was compromised by incubation in HBS-300 as well as seminal plasma. Moreover, there was an effect of the different treatments on sperm volumetric response to hypotonic challenge (Paper Ι). Given the deleterious effect of seminal plasma on caput sperm membrane integrity, the effects of different incubation systems on volume regulatory function (RVD) were considered only in cauda spermatozoa. The

sperm samples showed two volumetrically different subpopulations. No RVD was observed if seminal plasma was used as an incubation medium in either of these populations. The appearance, position and volume regulatory ability of the cells associated with these two different populations indicated that the exposure to the medium isotonic with seminal plasma was less beneficial than to the medium isotonic with the cauda epididymal plasma. It is concluded that recovery and incubation in a medium isotonic with caudal epididymal plasma (360 mOsm kg^-1) is a superior method for providing both caput and cauda epididymal spermatozoa for subsequent physiological studies, especially if their volume regulation ability is concerned. Very recently, Si and coworkers (2009) tested osmotic characteristics and fertility of murine sperm collected in different solutions. Spermatozoa collected in a hyperosmotic medium (isotonic with cauda epididymal plasma 415 mOsm kg^-1) resulted in an increase in sperm osmotic tolerance to higher osmolality compared with spermatozoa collected in isosmotic medium (hypotonic with cauda epididymal plasma 290mOsm kg^-1) and also resulted in high fertility of spermatozoa after cryopreservation (Si et al. 2009). These findings underline the present results (Paper Ι) and show the importance of the selection of the collection and incubation media for subsequent utilizations.

At the next stage, volume regulatory behaviour in caput and cauda epididymal spermatozoa was investigated. Two main subpopulations were observed in cell volume distributions of caput and cauda epididymal spermatozoa. Using electronic cell sizing, it was possible to determine the absolute cell volumes in physical units rather than as relative correlates of size, and to differentiate subpopulations of spermatozoa. In the studies on epididymal spermatozoa utilizing flow cytometry, such subpopulations related to different cell size, capable of responding to osmotic challenge and to undergo subsequent volume regulation were not detected (Yeung et al.

2002; 2004). It might be argued that the second sub-population in cauda epididymal spermatozoa samples is formed by immature spermatozoa bearing cytoplasmic droplets. Although virtually all caput spermatozoa have attached cytoplasmic droplets, they display also two clearly defined sub-populations. It therefore seems very unlikely that the second sub-population in cauda epididymal sperm simply represents spermatozoa with attached droplets. It is suggested that the second population represents swollen cells whose volume regulatory ability has been compromised in

some way. Such subpopulations of spermatozoa in ejaculated sperm have been observed in several species (Curry and Watson 1994; Kulkarni et al. 1997; Petrunkina et al. 2005a; Druart et al. 2009). In ejaculated sperm, appearance of subpopulations has been attributed mainly to uncontrolled ion uptake (Petrunkina et al. 2005b), or to possible interaction of transport mechanism and cytoskeleton at different ambient temperatures (Petrunkina et al. 2004a; 2004b).

Recently, Druat and coworkers hypothesized that these subpopulations might have different lipid compositions or they could be a result of different interactions of seminal plasma substances like proteins with different epididymal sperm subpopulations (Druart et al. 2009). Clearly, further investigations are needed on this point. medium 330 mOsm kg^-1, monkey: cauda epididymal fluid not assessed, recovery and incubation medium 290 mOsm kg^-1) (Yeung et al. 2002; 2004). In contrast, this study presents that in bull both caput and cauda epididymal spermatozoa display RVD after exposure to hypotonic challenge. Although there may be a species variation, another explanation may be differences in experimental approaches, such as volume measuring methods and choice of the experimental system. Druat et al. (2009) found that the hypotonic resistance, measured as the ratio of live/dead spermatozoa after a hypotonic stress markedly decreased during epididymal maturation and after ejaculation. Indeed, critical osmolality at which 50% of all spermatozoa died increased from 54.7 ± 3.2 to 116.7 ± 2.1 mOsm kg^-1 for the caput and cauda spermatozoa, respectively. The result indicates that caput spermatozoa are able to tolerate a more severe osmotic stress than cauda spermatozoa, which supports the findings of the present work (Paper I). We also found differences between caput and cauda spermatozoa, especially in terms of isotonic volumes of the subpopulations. The isotonic volume of caput spermatozoa was always higher compared to cauda spermatozoa. It decreased to the levels observed in cauda spermatozoa when in the recovery and

incubation media chloride and sodium were substituted by sulfate and choline. Furthermore, caput spermatozoa exhibit lower osmotic swelling after hypotonic stress compared to cauda spermatozoa, probably due to their already higher initial isotonic volume. Results of this study suggests that the increased volume of caput spermatozoa subpopulations relative to those in cauda samples results from poorly controlled sodium and chloride ion uptake from the external medium (Paper Ι). Inorganic (Na⁺, K⁺) and organic osmolytes (glutamate, myo-inositol and L-carnitine) are present in high concentrations in epididymal plasma (Crabo 1965; Casillas 1973;

Gustafsson et al. 1974; Cooper and Yeung 2003). During passage through the epididymis, the cytoplasm of maturing spermatozoa takes up many of these osmolytes (Cooper and Yeung 2003;

Yeung et al. 2006). Control of cell volume during uptake of these osmolytes would be facilitated by an ability to equilibrate ions freely across the plasma membrane channels. Subsequent closure of the channels in the mature cauda spermatozoa would ensure that volume control is maximized during the hypotonic stress occurring during ejaculation.

Previous studies by our group showed that tyrosine kinase activity is involved in closing volume-activated channels (Petrunkina et al. 2007a). The results of the present study indicate that tyrosine phosphorylation is lower in caput spermatozoa compared to cauda spermatozoa. The insufficient level of phosphorylation (possibly resulting from low tyrosine kinase activity or increased phosphatase activity) could be linked to the failure to closing a channel or to prevent premature activation of transport mechanisms favouring the uncontrolled ion uptake (Paper I).

Findings of Aitken and coworkers in mouse agree with this present result in the level of tyrosine phosphorylation of immature spermatozoa compared to mature spermatozoa (Aitken et al. 2007).

Another reason for differences between caput and cauda spermatozoa in their volume regulatory ability could partly be attributed to differences in lipid- and protein compositions of the plasma membrane, which are known to change during maturation in the epididymis (Nolan and Hammerstedt 1997; Kirchhoff 1998; Sullivan et al. 2007). Therefore, the effects of Fn2 proteins on the development of volume regulatory ability of spermatozoa during epididymal transit were examined in the second study (Paper ΙΙ). The long Fn2 proteins (ELSPBP1) are highly conserved

across the species, produced and secreted specifically by the epididymal duct epithelium and bound to spermatozoa. Hitherto, ELSPBP1 identified in the epididymis of a number of mammalian species e.g. human, dog, horse and pig but not in mice and rats (Saalmann et al.

2001; Schäfer et al. 2003; Ekhlasi-Hundrieser et al. 2005). In the present study it is shown that the long Fn2 protein (ELSPBP1) is present in the caput, corpus and cauda regions of the bull epididymis. It binds to the spermatozoa during their transit through the epididymis; it was detected in mature (cauda) spermatozoa but not in immature (caput) spermatozoa. Small Fn2 proteins (BSP proteins) bind to spermatozoa upon ejaculation (Nauc and Manjunath 2000). Both the long- and the small Fn2 proteins are localized in the head and midpiece regions of mature bovine spermatozoa. In vitro it was demonstrated that long Fn2 proteins bind phosphorylcholine head groups similar to small Fn2 proteins. The phospholipid-binding sites of small Fn2 proteins have been mapped to a pattern of invariant tryptophane and tyrosine residues which form the phosphorylcholine-binding pocket of each Fn2 module (Wah et al. 2002; Fan et al. 2006). The amino acids involved in lipid binding are strictly conserved in the small Fn2 proteins and in the first two Fn2 modules of the long variants (ELSPBP1). Furthermore, modeling of bovine long Fn2 protein indicates a comparable topology of secondary structure elements as shown for bovine BSP A1/2, suggesting a similar three-dimensional structure and ligand-binding ability (Paper ΙΙ, Fig. 4-5). As BSP-A1/2 is present in the seminal plasma in relatively large quantities, it was used as a model protein for Fn2 proteins in the present study. First BSP-A1/2 was isolated from a pool of seminal plasma obtained from fertile bulls, and then incubated with epididymal spermatozoa.

The binding of BSP-A1/2 to epididymal spermatozoa was confirmed by western blot analysis as well as by immunofluorescence microscopy. The results of BSP-A1/2 treatment were as following: two volumetrically different subpopulations in both caput and cauda spermatozoa existed after treatment. On the other hand, the treatment altered the ability of the lower-volume caput spermatozoa population to regulate their volume under hypotonic stress as compared with controls: initial swelling was higher and cell volumes were reduced after 20 min incubation. In other words, treatment with BSP-A1/2 enhanced the caput spermatozoa`s apparent ability to exhibit RVD. However, this apparent effect seems to be related rather to slowing down the accelerated volume regulation in caput sperm, likely to be caused by the premature activation of

ion channels/transport mechanisms. BSP-A1/2 appears to be involved in preventing or deactivating this premature activation. Cauda spermatozoa showed progressive volume regulation both in the absence and presence of BSP-A1/2 (Paper ΙΙ).

It is suggested that differences in volumetric behaviour between caput and cauda spermatozoa may be due to a lower degree of control of ion transport (K⁺, Cl^-). The structure and function of ion channels are sensitive to biophysical properties of their membrane microenvironment (e.g., fluidity, lateral pressure profile, and bilayer thickness) (Tillman and Cascio 2003). It is known thatbinding of BSP-A1/2 to membranes has an impact on membrane structure anddynamics. It causes a reduction of lipid mobility in plasma membrane of spermatozoa as well as in other lipid membranes (Müller et al. 1998; Ramakrishnan et al. 2001; Greube et al. 2004). Moreover, swelling-sensitive K⁺ and Cl^– channels are localized to the tail, midpiece and post-acrosomal region, very similar to the localization of the binding regions of the Fn2 proteins (Paper ΙΙ) (Barfield et al. 2005; Yeung et al. 2005). Hence, binding of BSP-A1/2 to phosphorylcholine headgroups in the plasma membrane could affect the swelling-activated channel activities via altered lipid-protein interactions. On the other hand, it may optimize their activities by reorganizing the distribution of the lipid rafts in which the signalling protein molecules are thought to be found, with the result that the activity of the signalling processes that control the swelling-activated channels are modulated (Tillman and Cascio 2003; Girouard et al. 2008). It seems highly probable that the attachment of ELSPBP1 to the maturing spermatozoa as they pass down the epididymis has the same eventual effect, whereby the mature (cauda) spermatozoa have the full ability to control their volume regulation.

In conclusion, recovery and incubation in a medium isotonic with caudal epididymal plasma (360 mOsm/kg) is a superior method for processing both caput and cauda epididymal spermatozoa for subsequent physiological studies. Caput epididymal spermatozoa are able to undergo RVD after hypotonic challenge. However, the mechanisms controlling ion transport under isotonic conditions are not fully developed compared to cauda epididymal (mature) sperm. It seems highly probable that the attachment of long Fn2 protein to the maturing spermatozoa has a

regulatory effect on cell volume control as they pass down the epididymis. For the first time, long Fn2 protein was detected in bovine epididymis and it was shown that it plays an important role in volume regulation of maturating epidiymal spermatozoa.

6. Summary

Evrim Sahin: Biochemical and physiological aspects of volume regulation in immature and mature bovine spermatozoa

Mammalian spermatozoa experience significant osmotic changes during their life span, both during ejaculation as well as during the transport in the female genital tract. Mature sperm possess a mechanism to maintain their volume and to regulate it after initial swelling following exposure to hypoosmotic conditions, named regulatory volume decrease (RVD).In rodents and primates RVD appears to be acquired during epididymal maturation, but in large domestic animals this aspect has not yet been examined. The aim of this study was to establish an optimal experimental system for physiological studies on cell volume of epididymal sperm and to investigate 1) whether bull epididymal spermatozoa are able to regulate their volume 2) whether this ability differs between caput and cauda epididymal spermatozoa and 3) whether bovine Fn2-module-containing proteins play a role in acquisition of volume regulation ability during sperm maturation. Sperm volumes were measured by electronic cell sizing; evaluation of plasma and acrosomal membranes integrities were performed using flow cytometry. It has been shown that the recovery and incubation in the medium isotonic with caudal epididymal plasma result in better viability and volume regulatory ability of epididymal spermatozoa than conventional system of media isotonic with seminal plasma, or seminal plasma itself. This experimental system was used for subsequent physiological studies. Two subpopulations were observed in volume distributions of caput and cauda epididymal spermatozoa, characterized by differing ability to swell in response to hypotonic conditions. Both sperm subpopulations in caput as well as in cauda were able to undergo RVD. Caput epididymal spermatozoa had a higher cell volume under isotonic conditions compared to cauda epidiymal spermatozoa. Substitution of sodium and chloride by choline and sulfate resulted in a decreased caput spermatozoa isotonic volume.

Furthermore, the differences abolished between caput and cauda epididymal spermatozoa. The results of the present study indicate that uptake of Na⁺ and Cl^- ions is less controlled in caput epididymal spermatozoa than in cauda, probably due to premature activation of transport mechanisms. Thus, although spermatozoa from the caput epididymidis are able to undergo RVD

after hypotonic challenge, the mechanisms controlling ion transport under isotonic conditions are not fully developed. To clarify this mechanism, the role of sperm binding Fn2-module-containing proteins was investigated. Mainly two classes exist which differ by having either two Fn2 modules, as the major member BSP-A1/2 (small Fn2 protein), or having four Fn2 modules such as ELSPBP1 (long Fn2 protein). The first two modules have similar three-dimensional structures.

Both proteins bind to the sperm through interaction with phosphorylcholine. It is shown that long Fn2 protein (ELSPBP1) is present in the caput, corpus and cauda regions of the bull epididymis whereas it binds to the spermatozoa during their transit through the epididymis; it was detected in mature (cauda) spermatozoa but not in immature (caput) epididymal spermatozoa. Small Fn2 proteins (BSP-A1/2) bind to spermatozoa upon ejaculation. Both the long and the small Fn2 proteins are essentially localized in the head and midpiece regions of mature bovine spermatozoa.

These common features suggest similar functions; therefore, BSP-A1/2 was used as a model protein. Cauda epididymal spermatozoa showed progressive volume regulation both in the absence and presence of BSP-A1/2. The protein treatment caused caput epididymal spermatozoa to swell more in response to hypotonic stress after 5 min and to regulate their volume after 20 min incubation in a similar fashion to cauda epididymal spermatozoa. It is suggested that BSP-A1/2 affects the premature activation of transport mechanisms (such as ion channels, for example). The premature activation of these channels could lead to uncontrolled uptake of Na⁺ and Cl^- ions. This uptake and coupled water transport is the likely cause of elevated isotonic volume levels in caput spermatozoa. At the same time, this premature activation accounts for the accelerated response to hypotonic challenge as the osmotically induced volume-sensitive response would be initiated faster. BSP-A1/2 possibly deactivates this premature activation.

Therefore, the response to hypotonic conditions becomes slower and the relative volumes in caput epididymal spermatozoa match those in cauda epiddiymal spermatozoa.

In conclusion, this study has provided further insights into the mechanism of spermatozoa volume regulation during epididymal maturation. A new superior experimental system was established for physiological volume regulation studies in bovine epididymal spermatozoa. Caput epididymal spermatozoa are able to undergo RVD after hypotonic challenge. However, the

mechanisms controlling ion transport under isotonic conditions are not fully developed compared to cauda epididymal (mature) sperm. It seems highly probable that the attachment of long Fn2 protein to the maturing spermatozoa has a regulatory effect on cell volume control as they pass down the epididymis. For the first time, long Fn2 protein was detected in bovine epididymis and its contribution to volume regulation of maturating epididymal spermatozoa is discussed.

7. Zusammenfassung

Evrim Sahin: Biochemische und physiologische Aspekte der Volumenregulation von unreifen und reifen bovinen Spermatozoen

Spermatozoen werden bei der Ejakulation und nach der Deponierung im weiblichen Genitaltrakt mit hypotonen Bedingungen konfrontiert, da Seminalplasma und uterine Flüssigkeit eine niedrigere Osmolalität aufweisen als die epididymale Flüssigkeit. Die hypotone Belastung führt zu einer primären Schwellung der Spermien. Reife Spermatozoen besitzen einen Mechanismus, um diese primäre Schwellung der Zelle wieder aufzuheben. Dieser Prozess der regulatorischen Volumenabnahme unter hypotonen Bedingungen wird als RVD („Regulatory Volume Decrease“) bezeichnet. Bei Nagetieren und Primaten wird vermutet, dass die Fähigkeit zum RVD während der Nebenhodenreifung erlangt wird; bei Nutztieren ist dieser Aspekt jedoch noch nicht hinreichend untersucht worden. In der vorliegenden Arbeit wurde ein optimiertes System für physiologische Studien bei Nebenhodenspermien etabliert, um die folgenden Fragen beantworten zu können: 1) Besitzen Nebenhodenspermien von Bullen die Fähigkeit zur Volumenregulation?

2) Unterscheidet sich die Fähigkeit zur Volumenregulation zwischen Nebenhodenkopf- und Nebenhodenschwanzspermien? 3) Sind bovine Fn2-Proteine an dem Erwerb der Volumenregulationsfähigkeit der Spermien beteiligt? Die Bestimmung des Zellvolumens erfolgte mit Hilfe des elektronischen Partikelzählers CASY 1; die Integritäten von Plasmamembran und Akrosom wurden durchflusszytometrisch ermittelt. Wurde für die Gewinnung und Inkubation der

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